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      An In Vitro TORC1 Kinase Assay That Recapitulates the Gtr-Independent Glutamine-Responsive TORC1 Activation Mechanism on Yeast Vacuoles

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          ABSTRACT

          Evolutionarily conserved target of rapamycin (TOR) complex 1 (TORC1) responds to nutrients, especially amino acids, to promote cell growth. In the yeast Saccharomyces cerevisiae, various nitrogen sources activate TORC1 with different efficiencies, although the mechanism remains elusive. Leucine, and perhaps other amino acids, was reported to activate TORC1 via the heterodimeric small GTPases Gtr1-Gtr2, the orthologues of the mammalian Rag GTPases. More recently, an alternative Gtr-independent TORC1 activation mechanism that may respond to glutamine was reported, although its molecular mechanism is not clear. In studying the nutrient-responsive TORC1 activation mechanism, the lack of an in vitro assay hinders associating particular nutrient compounds with the TORC1 activation status, whereas no in vitro assay that shows nutrient responsiveness has been reported. In this study, we have developed a new in vitro TORC1 kinase assay that reproduces, for the first time, the nutrient-responsive TORC1 activation. This in vitro TORC1 assay recapitulates the previously predicted Gtr-independent glutamine-responsive TORC1 activation mechanism. Using this system, we found that this mechanism specifically responds to l-glutamine, resides on the vacuolar membranes, and involves a previously uncharacterized Vps34-Vps15 phosphatidylinositol (PI) 3-kinase complex and the PI-3-phosphate [PI(3)P]-binding FYVE domain-containing vacuolar protein Pib2. Thus, this system was proved to be useful for dissecting the glutamine-responsive TORC1 activation mechanism.

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          Author and article information

          Journal
          Mol Cell Biol
          Mol. Cell. Biol
          mcb
          mcb
          MCB
          Molecular and Cellular Biology
          American Society for Microbiology (1752 N St., N.W., Washington, DC )
          0270-7306
          1098-5549
          8 May 2017
          29 June 2017
          15 July 2017
          : 37
          : 14
          Affiliations
          Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan
          Author notes
          Address correspondence to Tatsuya Maeda, maeda@ 123456iam.u-tokyo.ac.jp .

          Citation Tanigawa M, Maeda T. 2017. An in vitro TORC1 kinase assay that recapitulates the Gtr-independent glutamine-responsive TORC1 activation mechanism on yeast vacuoles. Mol Cell Biol 37:e00075-17. https://doi.org/10.1128/MCB.00075-17.

          Article
          PMC5492174 PMC5492174 5492174 00075-17
          10.1128/MCB.00075-17
          5492174
          28483912
          62612f5a-f32c-463c-8753-66039744cc34
          Copyright © 2017 American Society for Microbiology.
          Page count
          Figures: 7, Tables: 1, Equations: 0, References: 48, Pages: 16, Words: 9149
          Funding
          Funded by: Uehara Memorial Foundation https://doi.org/10.13039/100008732
          Award Recipient : Tatsuya Maeda
          Funded by: Noda Institute for Scientific Research (NISR) https://doi.org/10.13039/100010157
          Award Recipient : Tatsuya Maeda
          Funded by: MEXT | Japan Society for the Promotion of Science (JSPS) https://doi.org/10.13039/501100001691
          Award ID: 25291042
          Award ID: 17H03802
          Award Recipient : Tatsuya Maeda
          Categories
          Research Article
          Spotlight
          Custom metadata
          July 2017

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